CN109105736B - Method for producing grain fermentation enzyme powder with increased fermentation efficiency using liquid seed culture fluid inoculated with Bacillus coagulans strain - Google Patents

Method for producing grain fermentation enzyme powder with increased fermentation efficiency using liquid seed culture fluid inoculated with Bacillus coagulans strain Download PDF

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CN109105736B
CN109105736B CN201710693470.4A CN201710693470A CN109105736B CN 109105736 B CN109105736 B CN 109105736B CN 201710693470 A CN201710693470 A CN 201710693470A CN 109105736 B CN109105736 B CN 109105736B
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慎镛喆
朴哲
尹荣晟
金利洙
金雄镛
金爱香
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Abstract

The present invention relates to a method for producing a grain fermentation enzyme powder d having an increased fermentation efficiency using a liquid seed culture solution inoculated with a Bacillus coagulans strain, and to a grain fermentation enzyme powder produced by the method. The present invention provides a grain fermentation enzyme which can absorb and promote grain fermentation in a balanced manner and can supplement beneficial components, such as substrate rice bran, wheat bran, yeast extract, soybean powder, various essential nutrient components contained in isolated soybean protein powder, fermentation products generated during the fermentation of bacillus coagulans, digestive enzymes and various physiological active components, and can supplement diverse medicinal components and physiological active substances by fermenting grains and byproducts thereof through the bacillus coagulans. Moreover, the beneficial bacteria in the intestinal tract activate the intestinal function, the high-concentration amylase, protease and other components generated during fermentation effectively help to absorb the nutrient components contained in the grains and the food, and the fibrinolytic enzyme effectively dissolves thrombus and helps to circulate blood.

Description

Method for producing grain fermentation enzyme powder with increased fermentation efficiency using liquid seed culture fluid inoculated with Bacillus coagulans strain
Technical Field
The invention relates to a method for preparing cereal fermentation enzyme powder for increasing fermentation efficiency by utilizing a liquid seed culture solution inoculated with a bacillus coagulans strain, which specifically comprises the steps of (a) inoculating the bacillus coagulans strain into a substrate for seed culture, which is composed of rice bran, wheat bran, soybean powder, isolated soybean protein powder and yeast extract powder, so as to prepare the liquid seed culture solution; (b) cooking the grains after mixing with a culture substrate comprising rice bran, wheat bran, soybean powder, soybean protein isolate powder and yeast extract powder; (c) a step of adding the liquid inoculum culture solution produced in the step (a) to the grain cooked in the step (b) and fermenting the grain; (d) drying and powdering the fermented product formed by the fermentation in the step (c); the method for producing a cereal fermentation enzyme powder of (1).
Background
Enzymes are important proteins acting on metabolic activities in the body, and are substances that act as catalysts for chemical reactions. Specific categories include food enzymes that cannot be produced by themselves in the body and require ingestion in vitro, digestive enzymes that are produced by themselves in the body and facilitate digestion, and metabolic enzymes that act on metabolic activities other than digestion. Wherein the food enzyme is used for digestion and absorption, decomposition and excretion, anti-inflammatory and antibacterial, detoxication and sterilization, blood purification, cell revival and other physiological aspects. (currently used for enzyme therapy, p.29-41, 2013)
Enzymes are generally classified into digestive enzymes (amylase, protease, cellulase) which are involved in digestion, excretion, and nutrient intake in the human body, and metabolic enzymes (peripheral nerve, autonomic nerve, and various hormone regulation) which are involved in metabolism of nutrients ingested for life maintenance, and are mainly produced by culturing fermentation microorganisms. Fibrin aggregates platelets to form a blood clot, which gradually hardens and contracts to obstruct blood flow. The increase of fibrinolytic enzymes for decomposing fibrin in the body can effectively inhibit the aggregation of blood platelets and ensure the smooth blood flow.
The enzyme-containing food is a general term for adding a large amount of artificially cultured microorganisms to an edible material or extracting the enzyme components contained in the food or processing the enzyme components in order to enhance the functions of the enzymes. The enzyme-containing food contains various physiologically active substances generated by the fermentation and brewing processes of the enzyme and microorganism of the food, and various trace elements and physiologically active substances which are generated by the generation effect of nutrient components and the proliferation effect of beneficial bacteria and are beneficial to digestion and absorption. (Huh, S.H.and Kim, M.H.the modeln health and health food,1997, Hongkijjea press. Korea, p.35-36)
The bacillus coagulans is characterized by bacillus which produces lactic acid and lactic acid bacteria in addition to spores, is used as acid-resistant and heat-resistant lactic acid bacteria, produces various components and various physiologically active substances which are beneficial to human bodies, digestive enzymes, promotes physiological activities in the bodies and effectively promotes metabolism, and is beneficial to dyspepsia, allergic colon syndromes, constipation, cholesterol reduction and the like. Moreover, the components which are difficult to digest and the nutrient components in the grains are effectively decomposed into the nutrient components which are easy to be absorbed by the human body through fermentation.
In recent years, a method for producing various fermented foods by mixing brown rice and several kinds of main grains has been widely spread. Most of the hulls contain the major nutrients, but because the hulls are relatively hard and coarse, they are generally removed from the hulls for consumption. The rice bran and the wheat bran in the husk component are used as the substrate of the inoculum, so that the beneficial components in the husk can be effectively absorbed, and the beneficial components generated in the fermentation process can be absorbed at the same time.
Disclosure of Invention
[ problem ] to solve,
For this reason, the present inventors have selected excellent strains for developing a grain fermented powder that can be used as an enzyme-containing food through studies in the case, and have made this application.
It is an object of the present application to provide a process comprising,
(a) inoculating Bacillus coagulans strain into substrate composed of testa oryzae, testa Tritici, semen glycines powder, soybean protein isolate powder and yeast extract powder for inoculating bacteria to obtain liquid culture solution for inoculating bacteria;
(b) mixing rice bran, wheat bran, soybean powder, soybean protein isolate powder and yeast extract powder with the culture substrate, and steaming;
(c) a step of adding the liquid inoculum culture solution produced in the step (a) to the grain cooked in the step (b) and fermenting the grain;
(d) drying and powdering the fermented product formed by the fermentation in the step (c); the method for producing a cereal fermentation enzyme powder of (1).
It is another object of the present invention to provide a grain fermentation enzyme powder comprising one or more enzymes selected from the group consisting of amylase, protease, lipase, cellulolytic enzyme and cellulase, which are produced by the above method and have increased activity.
[ MEANS FOR SOLVING PROBLEMS ] A method for producing a semiconductor device
To achieve the object of the present invention, there is provided a liquid crystal display device including,
(a) inoculating Bacillus coagulans strain into substrate composed of testa oryzae, testa Tritici, semen glycines powder, soybean protein isolate powder and yeast extract powder for inoculating bacteria to obtain liquid culture solution for inoculating bacteria;
(b) mixing rice bran, wheat bran, soybean powder, soybean protein isolate powder and yeast extract powder with the culture substrate, and steaming;
(c) a step of adding the liquid inoculum culture solution produced in the step (a) to the grain cooked in the step (b) and fermenting the grain;
(d) drying and powdering the fermented product formed by the fermentation in the step (c); the method for producing a cereal fermentation enzyme powder of (1).
And a grain fermentation enzyme powder characterized by having an increased enzymatic activity, which is produced by the above-mentioned method, and which is selected from the group consisting of amylase, protease, lipase, fibrinolytic enzyme and cellulase.
The present invention is described in detail below.
The present invention provides a method for preparing a composite material comprising,
(a) inoculating Bacillus coagulans strain into substrate composed of testa oryzae, testa Tritici, semen glycines powder, soybean protein isolate powder and yeast extract powder for inoculating bacteria to obtain liquid culture solution for inoculating bacteria;
(b) mixing rice bran, wheat bran, soybean powder, soybean protein isolate powder and yeast extract powder with the culture substrate, and steaming;
(c) a step of adding the liquid inoculum culture solution produced in the step (a) to the grain cooked in the step (b) and fermenting the grain;
(d) drying and powdering the fermented product formed by the fermentation in the step (c); the method for producing a cereal fermentation enzyme powder of (1).
In the present invention, the step (a) is a step of preparing a liquid inoculum culture solution by inoculating a Bacillus coagulans strain into an inoculum substrate composed of rice bran, wheat bran, soybean powder, soybean protein isolate powder and yeast extract powder.
The substrate for the inoculum comprises soybean powder, rice bran, wheat bran, soybean protein isolate and yeast extract. Specifically, the substrate for the inoculum is prepared from 100 parts by weight of purified water, 1-5 parts by weight of soybean powder, 1-5 parts by weight of rice bran, 1-5 parts by weight of wheat bran, 1-5 parts by weight of soybean protein isolate and 0.1-3 parts by weight of yeast extract powder.
The substrate for the inoculum is inoculated with bacillus coagulans.
The Bacillus coagulans-inoculated strain is preferably a strain assigned to accession No. KCTC13284 BP. The strain is selected from traditional fermented food strains, and has the best starch, protein, fat and fibrin decomposition capability.
The inoculation is carried out by using a preculture solution for culturing the strain, using an isolated strain or using a strain powdered in a form such as freeze-drying or the like, or a culture of the strain. The inoculation amount of the bacillus coagulans strain is 5-20 parts by weight, preferably 10 parts by weight, of inoculation relative to 100 parts by weight of the mixture.
The culture solution for liquid inoculum is specifically liquid culture. The culture temperature is 30-40 ℃, and the culture time is 10-30 hours.
As demonstrated in the following examples, the seed substrate was inoculated to grains and cultured, and the protein in the grain fermentation enzyme obtained by the excellent activities of the starch decomposition enzyme, the lipase and the protease cellulase of the strain was reduced in molecular weight, thereby improving the digestibility and the activity of the fibrinolytic enzyme contributing to blood circulation.
The stage (b) is a stage of mixing rice bran, wheat bran, soybean powder, soybean protein isolate powder and yeast extract powder in the cereal, and then cooking the mixture.
The grain used in stage (b) is one of brown rice, soybean, barley, and mixed grains (wheat, corn, and coix seed). The grains are properly selected, cleaned and then soaked in clear water. And soaking the grains in clear water for 4-24 hours according to the types. The time of soaking in clean water can be adjusted within the range according to the actual characteristics of the grains. Specifically, the brown rice is 14-16 hours, the soybean is 5-9 hours, the barley is 3-5 hours, and the grains are mixed for 14-16 hours.
The soaking water temperature is 20-35 ℃, preferably 25-30 ℃. The reason for this is that a difference in moisture content occurs depending on a temperature difference during immersion. The washed and soaked grains are in a crushed form, or the grains are directly used without a crushing process.
The substrate for culture used in the stage (b) is formed by soybean powder, rice bran, wheat bran, soybean protein isolate and yeast extract powder. The mixture specifically comprises 100 parts by weight of the grain, 5-10 parts by weight of soybean powder, 1-5 parts by weight of rice bran, 1-5 parts by weight of wheat bran, 1-5 parts by weight of soybean protein isolate and 1-5 parts by weight of yeast extract powder.
The strain can be cultured from the grain in the non-cooking stage, but the heat treatment can kill the mixed bacteria in the grain, destroy the grain cell wall, gelatinize and deform the protein, provide an active breeding environment for microorganisms, and reduce the strain inoculation amount and the cost. The cooking may be performed using various methods disclosed in the industry, for example, using steam or superheated steam. The cooking is carried out in 2 stages. The stage 1 is a pre-cooking stage, which comprises a pre-cooking process at the temperature of 60-110 ℃ for 10-60 minutes, preferably 80-120 ℃ for 20-40 minutes. The 2 nd stage is the cooking stage, wherein the cooking is carried out at the temperature of 90-140 ℃ for 10-60 minutes, and preferably at the temperature of 100-120 ℃ for 30-50 minutes.
And in the cooking process, the water content is adjusted according to the expansion state of the grains for cooking. The grain in the original state at this stage is preferably 50 to 90% by weight of the whole grain, more preferably 60 to 80% by weight of the whole grain. In this stage, the air permeability is ensured by maintaining the original state of grains to form a space between grains.
After cooking, a cooling stage of the cooked cereal is added. After the cooking is finished, natural cooling can be carried out, or the cooling speed is increased to prevent overheating and balance cooling is carried out. The cooling temperature is specifically 35 ℃ to 50 ℃, and preferably 40 ℃ to 45 ℃. At temperatures below this range, the grain is too hard resulting in mixing difficulties.
The stage (c) is a stage of adding the liquid inoculum culture solution produced in the stage (a) to the grain cooked in the stage (b) and fermenting.
The amount of the liquid inoculum culture solution added is 5 to 20 parts by weight, preferably 10 parts by weight, based on 100 parts by weight of the grains contained in the mixture.
The culture method of the fermentation is not particularly limited, and a liquid culture tank, a rotary drum type fermenter, and a tray fermenter can be used. The fermentation of grains or a mixture thereof is facilitated in addition to the fermentation machine, and is not limited to the present application, and an appropriate apparatus may be selected for use according to production specifications. The culture temperature is 20-50 ℃ or 30-45 ℃, and the culture humidity is 40-90%, preferably 50-85%. The culture time is 1-72 hours, 12-72 hours, 36-50 hours or 48 hours.
And the stage (d) is a stage of drying the fermented product formed by the fermentation in the stage (c) and powdering the dried fermented product.
The drying stage can be carried out by various methods disclosed in the industry, but attention needs to be paid to drying in an environment with too high temperature, so that the bacteria in the grain fermentation product are killed, and the enzyme activity is reduced. Specifically, the strain is dried in an environment where the activity of the enzyme is maintained without death. The drying temperature is 30-60 ℃, preferably 35-45 ℃.
The drying time is 12 to 48 hours, preferably 20 to 28 hours. The water content of the dried fermentation product is 3-20%, preferably 3-7%. This is the optimal moisture content for the enzyme titer and preservation of the product.
The pulverization process is, according to the purpose of use of the grain fermented product, pulverization into various sizes, that is, crushing, grinding, milling, grinding, sanding, pulverizing, grinding or other means used when the food material is powdered or reduced into fine particles. Specifically, a hammer mill was used.
After the powdering process, the stage of randomly forming the powder is also included. Pulverizing fermented product, oven drying, and making into granule, pill, lozenge, or beverage.
The powdering or molding stage may be followed by a stage including packaging.
The present invention provides a grain fermentation enzyme powder characterized by having an increased enzymatic activity of at least one enzyme selected from the group consisting of amylase, protease, lipase, fibrinolytic enzyme and cellulase.
The present invention provides a thrombolytic food molding comprising the grain fermentation enzyme powder. The food molding is not particularly limited as long as it is a drinking food.
Specifically, the grain fermentation enzyme powder is food containing enzyme. The enzyme-containing food in the present invention means that, in order to strengthen the enzyme function, edible microorganisms are cultured in edible raw materials so that the materials contain a large amount of active enzymes; or extracting an enzyme-containing fraction from the food product; or enzyme-containing food products prepared by processing such food products.
The food molding of the present invention may include a health food molding. When the strain or the fermented cereal powder of the present invention is used as food or health food, the fermented cereal powder of the present invention may be added as it is or used together with other food or food ingredients, or may be used appropriately according to a usual method. The amount of the strain or grain fermentation product of the present invention to be mixed may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic prescription, etc.). The food molding is not particularly limited as long as it is an edible molding. The food-shaped product includes, for example, meat, sausage, bread, cake, chocolate, candy, jelly, snack, biscuit (cookie, biscuit, etc.), pizza, noodle (instant noodle, etc.), chewing gum, ice cream, etc. casein, various soups, tomato sauce, seasoning, gravy, sauce, beverage, tea, nutritional supplement, alcoholic drink, vitamin preparation, etc.
The food or health food molded product of the present invention may contain various flavors, natural carbohydrates, and other components, as in the case of ordinary beverages. The natural carbohydrate is monosaccharide such as glucose and fructose, disaccharide such as maltose and sucrose, polysaccharide such as dextrin and cyclodextrin, and sugar alcohol such as xylitol, sorbitol and erythritol. The sweetener can be natural sweetener such as thaumatin and stevia extract, or synthetic sweetener such as saccharin and aspartame. The natural carbohydrate is added in a ratio of each of the food or health food molded product of the present invention
Figure BDA0001378511390000061
0.01 to 0.20g, specifically about 0.04 to 0.10g, is added.
In addition to the above, the food or health food molding of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, PH regulators, stabilizers, preservatives, glycerin, alcohols, carbonating agents for carbonated beverages, and the like. In addition, the food or health food molding of the present invention may include pulp for producing natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in admixture. The addition ratio of these additives is selected within the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the food or health food molded product in the present invention.
Specifically, the molding for dissolving thrombus has a remarkable effect on treating or preventing myocardial infarction, thrombus, stroke, cerebral infarction, cerebral thrombosis or cerebral embolism.
The present inventors produced a grain fermentation enzyme powder having an increased enzyme content, and the specific enzyme content was as follows.
In the examples of the present invention, the enzyme activities of the culture solution for the liquid inoculum of grains and the grain fermentation enzyme powder to which the substrate for culture was added were measured. As a result, it was found that the activity of alpha-amylase was 3000-15000U/g, the activity of protease was 3000-15000U/g, the activity of lipase was 5-30U/g, and the activity of plasmin was 50-200U/g. This result showed a significant increase in enzyme activity compared to the grain without the substrate for cultivation.
[ Effect of the invention ]
The present invention provides a grain fermentation enzyme which can uniformly absorb and promote grain fermentation and supplement beneficial components such as substrate rice bran, wheat bran, yeast extract, soybean powder, various essential nutrient components contained in soybean protein isolate powder, fermentation products generated during the fermentation of bacillus coagulans, digestive enzymes and various physiological active components, and can supplement diversified medicinal components and physiological active substances by fermenting grains and byproducts thereof through the bacillus coagulans. Moreover, the beneficial bacteria in the intestinal tract activate the intestinal function, the high-concentration amylase, protease and other components generated during fermentation effectively help to absorb the nutrient components contained in the grains and the food, and the fibrinolytic enzyme effectively dissolves thrombus and helps to circulate blood.
Drawings
FIG. 1 is a schematic flow chart showing a method for producing a fermented enzyme powder of the present invention.
Detailed Description
The present invention is described in detail below. The embodiments are merely examples of the present invention and are not limited to the following.
< example 1>
< examples 1 to 1>
Method for measuring amylase activity
The test substance was contained in RO water (reverse osmosis water) at a concentration of 5g/100ml, and cultured with shaking at a temperature of 30 ℃ and a rated rotation speed of 200rpm for 30 minutes. The test chamber was spun at a rated speed of 12,000rpm for 3 minutes for circle center separation to obtain a supernatant, and the supernatant was filtered with a 0.45um filter. The filtrate was diluted appropriately with reverse osmosis water. For the empty test, the filtrate was deactivated by heating at 100 ℃ for 30 minutes. A glass test tube was prepared by placing 5ml of 1% soluble starch, 13ml of Mclvaine buffer, and 1ml of 0.1% calcium chloride. The substrate solution was pre-incubated at 37 ℃ for 10 minutes. 1ml of the sample solution or the empty sample solution was added thereto, and the reaction was carried out at 37 ℃ for 30 minutes. A15 ml cuvette was prepared with 10ml of iodine solution and 200ul of test/empty test reaction solution was added, and absorbance was measured at a wavelength of 660nm using distilled water as a control solution.
Figure BDA0001378511390000071
Definition of amylase titer Unit/g: the enzyme was allowed to react for 30 minutes at a titer contained in 1g of the test body, and a value of 1mg of starch was decomposed.
< examples 1 and 2>
Method for measuring protease activity
The test substance was contained in RO water (reverse osmosis water) at a concentration of 5g/100ml, and cultured with shaking at a temperature of 30 ℃ and a rated rotation speed of 200rpm for 30 minutes. The test chamber was spun at a rated speed of 12,000rpm for 3 minutes for circle center separation to obtain a supernatant, and the supernatant was filtered with a 0.45um filter. The filtrate was diluted appropriately with reverse osmosis water. 1ml of 0.6% casein was prepared in the test tube and preincubated at 37 ℃ for 3 minutes. 1ml of the sample solution or the empty sample solution was added thereto, and the reaction was carried out at 37 ℃ for 10 minutes. After adding 2ml of 0.4M trichloroacetic acid, the mixture was incubated at 37 ℃ for 25 minutes and then filtered through a 0.45um filter. 1ml of the filtrate was collected using a 15ml conical tube. After adding 5ml of 0.4M sodium carbonate and 1ml of the Fulin reagent to the liquid, incubation was carried out at 37 ℃ for 20 minutes and the absorbance was measured at a wavelength of 660 nm. After addition of 1ml of the test solution, the cells were incubated at 37 ℃ for 15 minutes. After 2ml of 0.4M trichloroacetic acid was added to the empty sample solution, 0.6% casein was immediately added thereto and mixed, and then the mixture was incubated at 37 ℃ for 25 minutes. Filter with a 0.45um filter. 1ml of the filtrate was collected using a 15ml conical tube. After adding 5ml of 0.4M sodium carbonate and 1ml of the Fulin reagent to the liquid, incubation was carried out at 37 ℃ for 20 minutes and the absorbance was measured at a wavelength of 660 nm.
Figure BDA0001378511390000081
Protease titer Unit/g is defined as the value calculated by converting the titer contained in 1g of the test body to the amount of L-tyrosine produced by the enzymatic reaction using the standard product.
< examples 1 to 3>
Method for measuring lipase activity
After diluting 5g of the test substance with glycine buffer, the solution was adjusted to 100ml with water. 0.05N sodium hydroxide solution was put into a dropper of a titration apparatus in advance, and the scale of the apparatus was adjusted to 30 ℃ and pH 7.0. 15.0ml of the base emulsion was placed in the reaction vessel of the titration apparatus, and a stirring magnet was placed therein. After a titrator was set on the reaction vessel, 1.0ml of a sample solution was added, and the titrator was started. The reaction was carried out by adjusting the pH to 7.0 using 0.05N sodium hydroxide solution, and a titration curve per minute was prepared at which 0.05N sodium hydroxide solution consumed ml.
(Note: 5 minutes after titration, the reaction shown in the recorder should be a straight line.)
The titer of the enzyme agent was calculated according to the following calculation formula.
Figure BDA0001378511390000082
R: titration consumption ml per minute of the Linear interval (ml/min)
N-regularity of sodium hydroxide solution
1,000: conversion of mmol of acid to. mu. mol
W amount of test sample contained in 1ml of sample solution (g)
Definition of potency: 1 enzyme Unit (LU) is the amount of enzyme that releases 1. mu. mol butyrate per minute from the substrate under the assay conditions.
< examples 1 to 4>
Method for measuring fibrinolytic enzyme activity
Test solution
Sodium borate buffer (50 mM): after 19.07g of sodium tetraborate and 9.0g of sodium chloride were dissolved in 900ml of distilled water, the pH was adjusted to 8.5 with hydrochloric acid, and distilled water was added to 1,000 ml.
Preparation of trichloroacetic acid solution (0.2M): trichloroacetic acid 32.678 was placed in distilled water and the amount was adjusted to 1,000 ml.
Fibrinogen solution (0.72%): after dissolving 96mg of fibrinogen (Sigma-aldrich, F8630) in 10ml of 50mM sodium borate buffer solution in a Erlenmeyer flask, the mixture was filtered using filter paper (Advantech No. 6). (modulation when using)
Preparing a thrombin solution: thrombin (Sigma-aldrich, T6634) was dissolved in 50mM sodium borate buffer, adjusted at a concentration of 1,000U/mL, and then a small amount of the resulting isolate was frozen in a microtube and stored. For the assay, the test solution was diluted 50-fold in 50mM sodium borate buffer.
Method of
Preparing a sample solution: the sample is diluted to a concentration of 0.67 to 1.33FU/g with 50mM sodium borate buffer. In 2 test tubes, 1.4ml of a sodium borate buffer solution and 0.4ml of a fibrinogen solution were collected, heated in a constant temperature water bath at 37 ℃ for 5 minutes, and then stirred with 0.1ml of thrombin. After the solution was placed in a constant temperature water bath AT 37 ℃ for 10 minutes, 0.1ml of the test solution was placed in 1 test tube (AT), stirred for 5 seconds and then left for 1 hour. (after leaving for 20 minutes every 5 minutes, stirring for 5 seconds) 0.2M trichloroacetic acid was added to the contents of 2 test tubes and stirred, and after the test tube (AB) in which no test solution was placed and stirred, the mixture was placed in a constant temperature water bath at 37 ℃ for 20 minutes. The solution was placed in a microtube and processed 5 minutes with 15,000x g circle center separations. The absorbance of 1ml of the supernatant was measured at a wavelength of 275 nm.
Calculation of
Fibrinolytic enzyme activity (FU/g) ═ AT-AB/0.01 x (1/60) x (1/0.1) x D
0.01 FU conversion factor (/ min)
Reaction time (min)
0.1 sample addition amount (mL)
Definition of potency: the 1-unit (1FU) is the number of enzymes which absorb light in an increased amount of 0.01 per minute at a wavelength of 275nm of the sample in the conventional test method.
< examples 1 to 5>
Method for measuring cellulase activity
Preparing a test solution: the final dilution of 1ml means that the test solution was diluted with water under the conditions of the test method so as to change the relative fluidity of the product within 5 minutes from 0.18 to 0.22. A certain amount of sample is put into a glass crusher and then added with water. After this was transferred to a measuring flask of appropriate capacity, an appropriate amount of water was added for dilution and filtration was performed with whatman No.1 or the like filter paper before use.
Experimental procedure: the viscometer with the corrected scale is fully soaked in detergent in advance, is cleaned by water and is vertically arranged in a glass water tank at the temperature of 40 ℃.20 mL of the base solution and 4mL of acetate buffer (pH 4.5) were collected and placed in a 50mL orbital Erlenmeyer flask, and 2 flasks for enzyme assay, each required for 1 sample, were prepared, with the base empty. The enzyme test flask was covered with a lid and placed in a water tank for 15 minutes, after the temperature was equilibrated, 1ml of a sample solution was collected and added to the flask, and the measurement time was started and the solution was sufficiently mixed. That is, 10ml of the mixed solution was collected, added to the main rod of the viscometer, and after waiting for 2 minutes, the rubber tube was connected to the fine rod of the viscometer, and the reaction solution was sucked up to the upper scale. For reaction times (T) within 15 minutesR) The reaction solution was repeatedly sucked up to the upper scale 4 times. The time until the solution reached the upper scale is measured and designated as TRAnd the time (seconds) from passing the upper scale to passing the lower scale is measured and set as TT. The reaction solution was sucked up to the upper scale from the start, and the time until the liquid surface reached the upper scale (minutes) was measured immediately) And is defined as TRThe time (seconds) from the upper scale to the lower scale is measured and determined as TT. A mixed solution of 20mL of the base solution and 4mL of acetate buffer (pH 4.5) was added to a flask for the base empty test, 1mL of water was added thereto, and after mixing, 10mL of the mixed solution was immediately collected and added to the main shaft of a viscometer, and the time T from the upper scale to the lower scale was repeatedly measuredSAfter 5 times (second), the mean value is taken as TS.10 ml of water, which is previously equilibrated at 40 ℃, is taken from a flask for a water-air test, a main rod of a viscometer is added, and the time T between reaching two scales is repeatedly measuredWAfter 5 times (second), the mean value is taken as TW. The 4 outflow times (T) are determined according to the following formulaT) And reaction time (T)R) Respectively corresponding relative fluidity (F)R) And TNThe value is obtained.
Figure BDA0001378511390000101
FR: relative flow for each reaction time
TS: average run-out time (seconds) according to the basal space test
TW: average outflow time (seconds) according to water control experiment
TTAccording to the average flow time (seconds) of the enzyme reaction solution
TRThe reaction elapsed time (minutes) (from the time of adding the sample solution to the time of starting measurement of the flow-out time (T)T) Chinese character zhi)
TNReaction time (T)R) (min) + test solution flow-out time (T)T) 1/2 (min)
Calibration lines were prepared using 4 relative flows corresponding to 4 reaction times. In this case, the line should be a straight line. The change in the line corresponds to a change in relative fluidity per minute, proportional to the amount of enzyme. The change in the line across the series of test points is more effective as a baseline for enzyme titer than the relative mobility value alone. F at 10 min and 5 min according to the calibration curveRThe value is obtained. The difference in flowability should be 0.18～0.22。
Figure BDA0001378511390000111
FR10: relative flow at 10 min reaction time
FR5: relative flow at 10 min reaction time
1,000: g is converted into mg
W: amount of sample (mg) contained in 1ml of sample solution
Definition of potency: 1 Cellulase Unit (CU) is the number of enzymes that produce a change in relative fluidity in the organocellulose substrate within 5 minutes under the test conditions.
Device
viscometer-A Scale corrected Canon-Fensk viscometer of size-100 or the same type is used.
-a glass water bath: a thermostatic water bath or the same type with a temperature of 40 ℃. + -. 0.1 ℃ is used.
Reagent and test solution
Acetate buffer (pH 4.5): 0.4N acetate buffer was added to 400ml of 0.4N acetic acid and the pH was adjusted to 4.5. + -. 0.05 with constant stirring.
-organic sodium cellulose: using organic sodium cellulose (cellulose gum Hercules (Aqualon) type 7HF or the same type)
-a base solution: the mixing vessel was filled with 200ml of water and stirred at a low speed. In order to prevent 1g of the dried organic cellulose sodium from being scattered outside the container, it was slowly dispersed in the container. After the inner wall of the vessel was washed with hot water using only a rubber stirring bar, the vessel was stirred at high speed for 1 minute with a lid. After transfer to a 500ml measuring flask, add water to an increment of 500ml and filter with gauze before use.
< example 2>
Method for measuring total bacteria number
1) And (3) test operation: after a test sample is uniformly mixed by using a sterilized glass rod and a sterilized reagent spoon, a certain amount (10-25 g) of the test sample is put into a sterilized container and mixed with 9 times of diluent to serve as a test solution. Respectively dripping 1ml of sterile sampling solution and 1ml of 10-fold stage diluent into more than 2 sterile culture dishes, maintaining the standard agar culture medium with the temperature of 43-45 ℃ for sterile division to be about 15ml, slowly and obliquely mixing the test body and the culture medium and solidifying. In order to inhibit the spread of colonies, 3-5 ml of standard agar medium is added again for overlapping. In this case, the time required for adding the culture medium after sampling the specimen should not exceed 20 minutes. After coagulation, the culture dish was placed upside down and cultured at 35. + -. 1 ℃ for 48. + -.2 hours (depending on the sample, it may be cultured at 30. + -. 1 ℃ or 35. + -. 1 ℃ for 72. + -.3 hours). The colony count was basically calculated by selecting a plate that had no colony spread and about 15 to 300 colonies were generated on 1 plate. 1ml of the same diluent without the addition of the test solution was used as a control sample to confirm whether the test was sterile or not.
2) And (3) calculating the colony number: immediately after the cultivation, the number of colonies generated was counted using a colony count computer. The special case is calculated within 24 hours after the preservation at the temperature of 5 ℃. The colony count was basically calculated by selecting a plate which had no colony spread (no problem when 1/2 or less was found overall) and in which about 15 to 300 colonies were formed on 1 plate. When more than 300 colonies were generated on the whole plate, the adjacent plates were counted according to the dense plate assay. When 15 or less colonies were formed on the whole plate, the dilution factor was the lowest and the measurement was performed
< example 1>
Selected strains
100g of brown rice, soybean, barley, and optionally one of mixed grains (wheat, corn, and coix seed) is prepared. The brown rice, soybean, and barley mixed grains (wheat, corn, and coix seed) are washed and sorted in advance. After the amount of water is prepared to completely soak the grain, the soaked 24 grain is moved to a tray. And putting the culture substrate 3, moving the tray into a cooking machine, cooking at 121 ℃ for 40 minutes, and cooling to 40-45 ℃ in the cooking machine. The trays were inoculated with Bacillus coagulans (KCTC13284BP), Bacillus amyloliquefaciens (KCTC 3002), Bacillus natto (KCTC 3239) and Lactobacillus fermentum (KCTC 13097) in a ratio of 10% of the amount of each grain. And (3) fermenting for 48 hours in an environment with the temperature of 30-40 ℃ and the humidity of 50-85%, drying and fermenting at the temperature of 40-60 ℃, and finally maintaining the water content of 3-7%.
The examples described above confirmed that Bacillus coagulans has the best activity against amylase, protease and lipase.
[ TABLE 1 ]
Setting of strains
Figure BDA0001378511390000121
Figure BDA0001378511390000131
< example 2>
Setting of substrate combination for inoculum
The method is carried out for determining the culture solution of the seed bacteria which is suitable for the best fermentation effect when the seed bacteria are used for the grains.
[ TABLE 2 ]
Base component for seed bacteria
Substrate for bacteria Combination (pure water 100 weight parts as benchmark)
Substrate 1 for seed bacteria 1% of soybean powder, 1% of rice bran, 1% of wheat bran, 1% of soybean protein isolate and 0.5% of yeast extract
Substrate 2 for seed bacteria 2% of soybean powder, 2% of rice bran, 2% of wheat bran, 1% of soybean protein isolate and 0.5% of yeast extract
Substrate 3 for seed bacteria Soybean powder 5%, rice bran 5%, wheat bran 5%, soybean protein isolate 1%, and yeast extract 0.5%
After producing a substrate for seed culture for liquid seed culture combination and sterilizing and cooling, Bacillus coagulans cultured in LB culture substrate (tryptone 1%, yeast extract 0.5%, sodium chloride 1%) was inoculated in an amount of 10% of the culture, and cultured at 37 ℃ and 180rpm for 15 hours to confirm the total number of bacteria.
First, 100g of brown rice, soybean, barley, mixed grains (wheat, corn, pearl barley) were prepared. The brown rice, soybean, and barley mixed grains (wheat, corn, and coix seed) are washed and sorted in advance. After the water quantity capable of completely soaking the grains is prepared, the grains are soaked according to different soaking time, and then the grains are moved to a tray. And moving the tray into a cooking machine, pre-cooking for 15 minutes at the temperature of 100 ℃, cooking for 30 minutes at the temperature of 121 ℃, and cooling to 40-45 ℃ in the cooking machine. Respectively putting the liquid culture solution for the inoculum substrates 1, 2 and 3 into 10% of the cooked and cooled grains, inoculating and mixing, fermenting for 48 hours in an environment with the temperature of 30-40 ℃ and the humidity of 50-85%, drying and fermenting at the temperature of 40-60 ℃, finally maintaining the moisture of 5-10%, crushing through 40 sieve meshes, and determining the total number of bacteria.
The total number of bacteria in the liquid inoculum culture solution and the final cereal fermentation enzyme powder was confirmed, and it was found that the total number of bacteria in the inoculum base 2 and the inoculum base 3 was the largest, and the inoculum base 2 was set to the most economically feasible and efficient combination. (see Table 3)
[ TABLE 3 ]
Final powder total bacteria count of liquid strain culture liquid
Figure BDA0001378511390000141
< example 3>
Setting of soaking time
To determine the soaking time corresponding to the optimum fermentation conditions of the grains in the examples, 100g of grains were soaked for 4 hours, 7 hours, 15 hours, and 24 hours, respectively, after the amount of water was first prepared to completely soak the grains. And (3) transferring the soaked grains to a tray, putting a culture substrate into the tray, transferring the tray into a cooking machine, cooking for 40 minutes at the temperature of 121 ℃, and cooling to 40-45 ℃ in the cooking machine. After cooling, the trays are respectively inoculated with culture bacteria of solidified bacillus which are used for comparing the grains with the same bacteria number of 10 percent of the weight in the precooking time. And (3) fermenting for 48 hours in an environment with the temperature of 30-40 ℃ and the humidity of 50-85%, drying and fermenting at the temperature of 40-60 ℃, and finally maintaining the water content of 3-7%.
The examples show that the activities of amylase, protease and lipase are most preferable when soybeans are soaked for 7 hours, barley is soaked for 4 hours, brown rice is soaked for 15 hours, and mixed grains (corn, wheat and coix seeds) are soaked for 15 hours. (refer to Table 4)
[ TABLE 4 ]
Setting of soaking time
Figure BDA0001378511390000142
Figure BDA0001378511390000151
< example 4>
Setting of precooking time
In order to determine the cooking time corresponding to the optimum fermentation conditions of grains, 100g of grains are respectively soaked after the water amount capable of completely soaking the grains is prepared. Soaking semen glycines for 7 hr, soaking fructus Hordei vulgaris for 4 hr, soaking brown rice for 15 hr, soaking mixed grains (semen Maydis, semen Tritici Aestivi, and Coicis semen) for 15 hr, and transferring to a tray. After the substrate for culture is put into the pot, the pot is moved to a cooking machine and is pre-cooked for 10 minutes, 30 minutes and 60 minutes at the high temperature of 80 ℃, and is pre-cooked for 10 minutes, 30 minutes and 60 minutes at the high temperature of 100 ℃, and is formally cooked for 30 minutes at the high temperature of 121 ℃ and then is cooled to 40-45 ℃ in the cooking machine. After cooling, the pre-cooking time is respectively compared with the 10 percent of the amount of the culture bacteria of the same bacteria number, namely the bacillus coagulans, in the trays. Fermenting for 48 hours in an environment with the temperature of 30-40 ℃ and the humidity of 50-85%, drying and fermenting at the temperature of 40-60 ℃, and finally maintaining the water content of 3-7%.
The examples show that the activities of amylase, protease and lipase were optimal when precooking was carried out at 100 ℃ for 30 minutes. (refer to Table 5)
[ TABLE 5 ]
Setting of precooking time
Figure BDA0001378511390000152
Figure BDA0001378511390000161
< example 5>
Setting of fermentation conditions
In order to set the optimal fermentation conditions for grains, the enzyme titer is determined by fermenting the brown rice for 12 hours, 24 hours, 36 hours, 48 hours, 60 hours and 72 hours under the conditions that the fermentation temperature is respectively 30 ℃,37 ℃ and 40 ℃, and the fermentation temperature is respectively 50%, 60%, 70% and 85%.
By the examples, the activities of amylase, protease and lipase were optimized when fermented at 37 ℃ and 70% moisture for 48 hours. (see tables 6, 7 and 8.)
[ TABLE 6 ]
Setting of fermentation temperature
Figure BDA0001378511390000162
[ TABLE 7 ]
Setting of fermentation humidity
Figure BDA0001378511390000163
[ TABLE 8 ]
Setting of fermentation time
Figure BDA0001378511390000171
< example 6>
Setting of fermentation drying conditions
In order to set the optimal fermentation conditions for grains, after brown rice and soybeans are fermented, the brown rice and soybeans are respectively dried for 12 hours, 24 hours and 48 hours at the drying temperatures of 30 ℃,40 ℃, 50 ℃ and 60 ℃ to confirm the enzyme titer and the water content.
Through the embodiment, when the mixture is dried and fermented at 40 ℃ for 24 hours, the optimal moisture content of 5 percent and two conditions of amylase, protease and lipase activity can be simultaneously met. (refer to Table 9)
[ TABLE 9 ]
Figure BDA0001378511390000172
Figure BDA0001378511390000181
< example 7>
Setting of the present substrate combination for culture
100g of brown rice, soybean, barley, and mixed grains (wheat, corn, and coix seed) are prepared. The brown rice, soybean, and barley mixed grains (wheat, corn, and coix seed) are washed and sorted in advance. After preparing the water amount capable of completely soaking the grains, soaking the grains according to the different soaking time, and then transferring the grains to a tray.
[ TABLE 10 ]
Substrate assembly for culture
Substrate for culture Combination (pure water 100 weight parts as benchmark)
Substrate for culture 1 Soybean powder 5%, rice bran 5%, wheat bran 5%, soybean protein isolate 1%, and yeast extract 1%
Substrate for culture 2 10% of soybean powder, 5% of rice bran, 5% of wheat bran, 1% of soybean protein isolate and 1% of yeast extract
Substrate for culture 3 10% of soybean powder, 5% of rice bran, 5% of wheat bran, 2% of soybean protein isolate and 1% of yeast extract
Substrate for culture 4 10% of soybean powder, 5% of rice bran, 5% of wheat bran, 5% of soybean protein isolate and 2% of yeast extract
100 parts by weight of grain are added and mixed to the substrate for non-additive culture and the substrates 1 to 4 of the culture solution, respectively. And (3) moving the tray to a cooking machine, pre-cooking for 15 minutes at the high temperature of 100 ℃, cooking for 30 minutes at the high temperature of 121 ℃, and cooling to 40-45 ℃ in the cooking machine. A 10% aliquot of culture substrate was added to the cereal in the cereal tray versus a 10% aliquot. After mixing, maintaining the temperature at 30-40 ℃ and the humidity at 50-85%, fermenting in a fermentation chamber for 48 hours, drying and fermenting at the temperature of 40-60 ℃, and finally maintaining the water content at 3-7%.
The total number of bacteria and the enzymatic activity of the final cereal fermentation enzyme powder other than the culture substrate were confirmed, and it was found that the total number of bacteria was significantly larger when the culture substrate was added and the enzymatic activity was more prominent than when the culture substrate was not added. (refer to Table 11)
[ TABLE 11 ]
Enzyme activity capability based on culture substrate
Figure BDA0001378511390000182
Figure BDA0001378511390000191
[ possibility of Industrial utilization ]
The invention can supplement various medicinal components and physiological active substances by fermenting grains and bacillus coagulans serving as byproducts thereof. Moreover, the invention has the effect of activating the intestinal function by beneficial bacteria in the intestinal tract, being beneficial to absorbing the nutritional ingredients contained in grains and foods such as high-concentration amylase, protease, lipase and the like generated during fermentation, and the effect of dissolving thrombus by using the fibrinolytic enzyme and being beneficial to blood circulation, and is the invention with industrial utilization possibility.
[ preservation information ]
The name of the depository: korean type culture Collection
Address: korea Arbor
And (3) classification and naming: bacillus coagulans

Claims (13)

1. A method for producing a cereal fermentation enzyme powder, comprising:
(a) inoculating Bacillus coagulans strain into substrate for bacteria culture composed of testa oryzae, testa Tritici, semen glycines powder, soybean protein isolate powder and yeast extract powder to obtain liquid culture solution for bacteria culture;
(b) mixing rice bran, wheat bran, soybean powder, soybean protein isolate powder and yeast extract powder with the culture substrate, and steaming;
(c) a step of adding the liquid inoculum culture solution produced in the step (a) to the grain cooked in the step (b) and fermenting the same;
(d) drying and powdering the fermented product formed by the fermentation in the step (c);
the collection number of the bacillus coagulans strain is KCTC13284 BP.
2. A method for producing a cereal fermentation enzyme food, comprising:
(a) inoculating Bacillus coagulans strain into substrate for bacteria culture composed of testa oryzae, testa Tritici, semen glycines powder, soybean protein isolate powder and yeast extract powder to obtain liquid culture solution for bacteria culture;
(b) mixing rice bran, wheat bran, soybean powder, soybean protein isolate powder and yeast extract powder with the culture substrate, and steaming;
(c) a step of adding the liquid inoculum culture solution produced in the step (a) to the grain cooked in the step (b) and fermenting the same;
(d) drying and powdering the fermented product formed by the fermentation in the step (c);
(e) converting the powder of stage (d) into beverage, granule, powder pill, capsule forming stage;
the collection number of the bacillus coagulans strain is KCTC13284 BP.
3. The method according to claim 1 or 2, wherein the grain in the stage (b) is selected from one or more of the group consisting of brown rice, barley, soybean, and mixed grains composed of corn, wheat, and pearl barley.
4. The method according to claim 1 or 2, wherein the grain in the stage (b) is soaked for 3 to 16 hours.
5. The method according to claim 1 or 2, wherein the substrate for the inoculum in the stage (a) is selected from the group consisting of 100 parts by weight of purified water, 1 to 5 parts by weight of soybean powder, 1 to 5 parts by weight of rice bran, 1 to 5 parts by weight of wheat bran, 1 to 5 parts by weight of soybean protein isolate, and 0.1 to 3 parts by weight of yeast extract powder.
6. The method according to claim 1 or 2, wherein the culture substrate in the stage (b) comprises 100 parts by weight of the grain, 5 to 10 parts by weight of the soybean powder, 1 to 5 parts by weight of the rice bran, 1 to 5 parts by weight of the wheat bran, 1 to 5 parts by weight of the soybean protein isolate, and 1 to 5 parts by weight of the yeast extract powder.
7. The manufacturing method according to claim 1 or 2, wherein the cooking in the stage (b) is performed at a high temperature of 70 ℃ to 140 ℃ for 10 to 60 minutes.
8. The manufacturing method according to claim 1 or 2, wherein the cooking in the stage (b) comprises precooking at a high temperature of 60 ℃ to 110 ℃ for 10 to 60 minutes.
9. The manufacturing process according to claim 1 or 2, characterized in that the fermentation in stage (c) is carried out at a temperature ranging from 30 ℃ to 40 ℃ or at a humidity ranging from 50% to 85%.
10. The manufacturing method according to claim 1 or 2, wherein the drying in the step (d) is performed at a temperature of 35 ℃ to 45 ℃ for 20 to 28 hours.
11. The method according to claim 1 or 2, wherein the liquid inoculum culture solution of the stage (c) is added in an amount of 5 to 20 parts by weight based on 100 parts by weight of the grain of the stage (b).
12. The cereal enzyme powder produced by the method according to claim 1, wherein the cereal fermentation enzyme powder has an increased enzymatic activity of one or more enzymes selected from the group consisting of amylase, protease, lipase, fibrinolytic enzyme and cellulase.
13. A finished food product for improving digestion comprising the grain fermentation enzyme powder of claim 12.
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